Tumor cells having tumorigenic potential but lacking invasive/metastatic potential, method for preparing them and screening method for metastasis-related genes using the same

ABSTRACT

Tumor cells having tumorigenic potential but lacking invasive/metastatic potential are established by introducing an oncogene of the ras family into a BALB/c 3T3 A31-variant cell. A screening method for genes having the property of conferring invasive/metastatic potential is also provided, which comprises transfecting DNA derived from a tumor tissue obtained from the surface or inside of a mammal or derived from a tumor cell line into tumor cells having tumorigenic potential but lacking invasive/metastatic potential, isolating cells having acquired invasive/metastatic potential and extracting DNA therefrom.

FIELD OF THE INVENTION

The present invention relates to tumor cells having tumorigenicpotential but lacking invasive/metastatic potential, a method forpreparing them and a screening method for metastasis-related genes usingthem.

PRIOR ART

Invasion and metastasis occur at the final stage of tumor progressionand are the major cause of death of cancer patients. However, it isdifficult to treat such effects because the molecular mechanism by whichmalignant phenotypes of tumor cells such as invasion/metastasis areexpressed has been mostly unknown. Although the expression of malignantphenotypes such as invasion/metastasis has long been recognized as abiological phenomenon which is pathologically distinguished from tumorformation in carcinogenic process, there has been only limitedinformation about biochemical basis therefor and target groups ofmolecules for therapy have not been well identified.

Generally, accumulation of alterations of genetic information ofoncogenes and tumor suppressor genes is thought to make cells cancerousand sometimes invasive/metastatic. Namely, a gene that plays a role inmediating an external signal into the nucleus is mutated to form a geneproduct, which activates the downstream signaling cascade continuously.This causes phenotypes of tumor cells to be expressed. It is alsothought that tumor cells become invasive/metastatic during tumorprogression as a result of additional genetic alterations to thoserequired for tumorigenicity (Evans C. W. The Metastatic Cell: Behaviorand Biochemistry, Chapman & Hall, London, 1991). Therefore, metastasisof tumor cells may be induced by an aberrant intracellular signalingsystem that is different from the signaling system required fortumor-igenicity. Thus, it is very important for investigations of themechanism of invasion/metastasis to elucidate a metastasis-specifictumor-related signaling pathway that is responsible for tumor cellsbecoming invasive/metastatic.

For such investigations, a cell which could separably acquiretumorigenic potential and invasive/metastatic potential by oncogenetransfer would be useful for identifying the above pathway and moleculesinvolved therein.

A set of two cells derived from the same parent strain, one of which hastumorigenic potential but lacks invasive/metastatic potential while theother has both potentials, would be useful for investigating thesignaling pathway involved in metastasis. Some tumor cell linessatisfying this criterion have already been established. They includerodent-derived cell lines K1735, B16, T-lymphoma L5178Y, Lewis lungcarcinoma and rat ascites hepatoma AH7974, as well as their derivativesobtained by in vitro culture and selection of cells recovered from invivo metastases of these cell lines (Fidler I. J. et al., J. Natl.Cancer Inst. 67:947-956, 1981; Talmadge J. E. et al., J. Natl. CancerInst. 69:975-980, 1982; Fidler I. J., Nature 242:148-149, 1973; FidlerI. J., Cancer Res. 35:218-224, 1975; Fidler I. J. et al., Am. J. Pathol.97:633-648, 1979; Ota et al., Clin. Exp. Metastasis 10:297-308, 1992;Olsson L. et al., Cancer Res. 41:4706-4709, 1981; Brodt P., Cancer Res.46:2442-2448, 1986; Pal. K. et al., Invasion Metastasis 5:159-169, 1985;Young M. R. et al., Cancer Res. 45:3918-3923, 1985; Kawaguchi T. et al.,Clin. Exp. Metastasis 10:225-238, 1992). However, phenotypes of thesecell lines are sometimes unstable during experimentations (Poste G. etal., Proc. Natl. Acad. Sci. USA 78:6226-6230, 1981) and many propertiesof these cell lines that are not related to the acquisition ofinvasive/metastatic potential may change during in vivo selection.

It would be useful and valuable if a stable set of cell lines asdescribed above could be obtained by oncogene transfer. However, whetheror not cells acquire tumorigenic potential and invasive/metastaticpotential by oncogene transfer depends on the nature of the cell lineused (Muschel R. J. et al., Am. J. Pathol. 121:1-8, 1985). Moreover,oncogene transfer frequently induces invasive/metastatic potential inrecipient cells to a greater or lesser extent simultaneously withtumorigenicity (Thorgeirsson U. P., Mol. Cell. Biol. 5:259-262, 1985;Egan S. E. et al., Science 238:202-205, 1987). Up to the present, noreport has shown exact establishment of a cell line having tumorigenicpotential but lacking invasive/metastatic potential and a cell linehaving both of these potentials. Thus, it was not easy to separately usecharacteristics of tumorigenic potential and invasive/metastaticpotential of tumor cells for investigations in an experimental system.

It is an object of the present invention to establish a tumor cell linehaving tumorigenic potential but lacking invasive/metastatic potential,which is suitable for investigating the difference between the signalingpathway for acquiring tumorigenic potential and the signaling pathwayfor acquiring invasive/metastatic potential. Such a tumor cell line canbe used in combination with a cell line derived from the same parentstrain but having both of tumorigenic and invasive/metastatic potentialsfor investigations of characteristics of invasive/metastatic potential.It is another object of the present invention to develop a screeningmethod for tumor metastasis-related genes using the tumor cell line ofthe present invention.

SUMMARY OF THE INVENTION

As a result of careful studies to overcome the above problems, wesucceeded in establishing a tumor cell line having tumorigenic potentialbut lacking invasive/metastatic potential by introducing a specificoncogene into a recipient cell.

Accordingly, the present invention provides a tumor cell havingtumorigenic potential but lacking invasive/metastatic potential. Apreferred tumor cell of the present invention having such properties is1-1ras1000 deposited with the National Institute of Bioscience andHuman-Technology of the Agency of Industrial Science and Technologyunder accession number FERM BP-5406.

The deposit was made on Feb. 20, 1996 and the address of the NationalInstitute of Bioscience and Human Technology Agency of IndustrialScience and Technology, Ministry of International Trade and Industry, is1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan.

The present invention also provides a method for preparing said tumorcell having tumorigenic potential but lacking invasive/metastaticpotential by introducing an oncogene of the ras family into a BALB/c 3T3A31 variant cell.

The present invention also provides a screening method for genes havingthe property of conferring invasive/metastatic potential, whichcomprises transfecting DNA derived from a tumor tissue obtained from thesurface or inside of a mammal or derived from a tumor cell line into atumor cell having tumorigenic potential but lacking invasive/metastaticpotential, isolating cells having acquired invasive/metastatic potentialand extracting DNA therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The copy number of introduced mutated c-Ha-ras gene by slot blothybridization in ras-transformed A31 variant cells (electrophoreticphotographs).

FIG. 2. Immunoblotting analysis of ras-transformed A31 variant cellsusing an anti-p21^(ras) monoclonal antibody NCC-RAS-004 (electrophoreticphotographs). Lane 1:5 ng of standard protein p21Gly-12 corresponding tothe product of normal human c-Ha-ras1 plus 5 μl of lysate from T24bladder carcinoma cells carrying mutated c-Ha-rasl gene; Lane 2:5 μl oflysate from A31-1-1 cells: Lane 3:50 μl (10-fold excess) of lysate fromA31-1-13 cells; Lane 4:5 μl of lysate from A31-1-1ras cells; Lane 5:5 μlof lysate from 1-1ras1000 cells; Lane 6:5 μl of lysate from A31-1-13cells; Lane 7:5 μl of lysate from 1-13ras1000 cells.

FIG. 3. (Parts A-B) Immunoblotting analysis (A) and phosphorylationassay (B) of v-src-transformed BALB/c 3T3 A31 variant cells(electrophoretic photographs). Lane 1: 1-1; Lane 2:1-13; Lanes3-6:1-1-derived src-transformants; Lanes 7-10:1-13-derivedsrc-transformants.

FIG. 4. Invasive or metastatic potentials of ras-transformed 1-1ras1000and src-transformed 1-1src cells in either wild-type or NK-suppressedBALB/c mice (morphologic photographs the organisms).

FIG. 5. (Parts A-B) In vitro invasion assayed by counting the number ofcells penetrating MATRIGEL-coated filters using a microscope of 400magnifications (A), and cell motility as the motility index measured bycomputer-aided digital image analysis (B). Each column indicates themean of triplicate samples with standard deviation. Column 1:1-ras;Column 2: 1-1ras1000; Column 3:1-13ras; Column 4:1-13ras1000; Column5:1-1src; Column 6:1-13src.

DETAILED DESCRIPTION OF THE INVENTION

Suitable recipient cells for preparing a cell line having tumorigenicpotential but lacking invasive/metastatic potential may preferably beselected so that the resulting cell line can be used to prepare acounterpart of the above set of cell lines, i.e. a cell line having bothof tumorigenic potential and invasive/metastatic potential. In thisrespect, suitable recipient cells for oncogene transfer should have thefollowing properties:

1) they should have a stable non-metastatic phenotype;

2) they should be able to afford a cell which has acquiredinvasive/metastatic potential by oncogene transfer;

3) they should be able to select a cell which has acquiredinvasive/metastatic potential by oncogene transfer.

Recipient cells satisfying these properties include but are not limitedto, for example, BALB/c 3T3 A31 cells (Kakunaga T., et al. (eds.)Transformation Assay of Established Cell Lines: Mechanisms andApplication, Oxford University Press, New York, 1985, pp. 55-73), andother cells having the above properties. BALB/c 3T3 A31 cells include,for example, BALB/c 3T3 A31-1-1, BALB/c 3T3 A31-1-8 and BALB/c 3T3A31-1-13 established by Kakunaga as used in the examples of the presentinvention (Kakunaga et al., Science 209:505-507, 1980).

The oncogene to be introduced into a recipient cell may be an oncogenewhich allows the recipient cell to form a focus (focus formation or aswell of cells means cancerization of cells) and has the activity ofconferring no invasive/metastatic potential, such as a gene of the rasfamily. According to the present invention, tumor cells havingtumorigenic potential but lacking invasive/metastatic potential could beestablished particularly by using, but not limited to, the activatedc-Ha-ras.

Tumor cells of the present invention are prepared by providing a plasmidcarrying said oncogene and introducing it into a recipient cell. Aconvenient method for introducing an oncogene into a recipient cell iselectroporation (Tatsuka et al., Exp. Cell Res. 178:154-162, 1988), butother means such as the calcium phosphate method, microinjection mayalso be used.

Transformed cells are seeded in dishes to assay focus formation, andarising transformed foci are isolated and multiplied as an independentcell strain or pooled.

Correct integration of the oncogene in the transformed cells can beascertained by Southern blot analysis, while transcription of theintegrated gene into mRNA can be ascertained by Northern blot analysisof the transcribed mRNA. Furthermore, translation of mRNA into proteincan be ascertained by immunoblotting analysis using, for example, ananti-p21^(ras) antibody.

An animal is used to evaluate tumorigenic potential andinvasive/metastatic potential of thus obtained cell line. In the presentinvention, BALB/c nude mice were used. In tumorigenicity assay,transformed cells were subcutaneously injected into the nude mice toobserve whether or not a tumor is formed, and growth rate and latencyperiod (the period from injection to detection of a tumorous nodule). Inexperimental metastasis assay, transformed cells were injected into thetail vein of the nude mice, and after 5 weeks, the mice were sacrificedand autopsied for metastasis.

The present invention thus succeeded in establishing cell lines havingtumorigenic potential but lacking invasive/metastatic potential asdescribed above. A particularly preferable cell line established by thepresent invention was designated as 1-1ras1000 and internationallydeposited with the National Institute of Bioscience and Human-Technologyof the Agency of Industrial Science and Technology (residing at 1-3,Higashi 1-Chome, Tsukuba-city, Ibaraki-prefecture, 305 Japan) on Feb.20, 1996 under accession number FERM BP-5406. 1-13ras1000 is anotherpreferable tumor cell line having similar properties to those of1-1ras1000.

1-1ras1000 is a clone obtained by amplification to enhance theexpression level of the oncogene introduced, in which the ras gene isoverexpressed at about 16-fold amplification of the parent strainisolated from foci. The non-metastatic phenotype of 1-1ras1000 cells isso stable that they can not be turned metastatic even after in vitropassage or treatment with initiators such as UV rays ormethylcholanthrene or treatment with TPA. It is very difficult to obtainan invasive/metastatic clone from these cells by selection involvingpermeation through micropore filters coated with basement membrane(MATRIGEL). MATRIGEL is a basement membrane matrix extracted from theEngelbreth-Holm-Swarm mouse tumor and is rich in basement membraneproteins. The major matrix components are laminin, collagen IV,entactin, and heparan sulfate proteoglycan (perlecan); the matrix alsocontains growth factors, matrix metalloproteinases and otherproteinases.

Thus, 1-1ras1000 cells have a very stable non-metastatic phenotypewithout expressing metastatic phenotype. However, it is possible thatthis property may result from potential lack of invasive/metastaticpotential in 1-1ras1000 cells. The src oncogene introduced into thesecells induced a great number of metastases in lung. This proved that1-1ras1000 cells do not lack the step of acquiring invasive/metastaticpotential. The cells that took up invasive/metastatic src showed ahigher motility than the cells that took up ras on basement membrane(MATRIGEL). Also in experiments using micropore filters, src-transformedcells were invasive for basement membrane.

The present invention thus provides a set of a tumor cell line havingtumorigenic potential but lacking invasive/metastatic potential and atumor cell line having both of tumorigenic potential andinvasive/metastatic potential as described above, allowingcharacteristics of invasive/metastatic potential and tumorigenicpotential to be separately used for investigations.

Cell lines having tumorigenic potential but lacking invasive/metastaticpotential of the present invention can be used for screening for genesconferring invasive/metastatic potential. The screening method for geneshaving the property of conferring invasive/metastatic potentialaccording to the present invention can be performed by transfecting DNAderived from a tumor tissue obtained from the surface or inside of amammal or derived from a tumor cell line into a cell line of the presentinvention, isolating cells having acquired invasive/metastatic potentialand extracting DNA therefrom.

A specific example of the screening method for genes having the propertyof conferring invasive/metastatic potential using strain 1-1ras1000 ofthe present invention can be performed by the following procedures:

1) Prepare a genomic gene of a metastatic tumor cell (derived from atumor tissue obtained from the surface or inside of a mammal or derivedfrom a tumor cell line) or a cDNA library (derived from a tumor tissueobtained from the surface or inside of a mammal or derived from a tumorcell line) constructed with a mammalian cell expression vector.

2) Introduce either the genomic gene or cDNA library obtained in 1) anda selectable marker (for example, a hygromycin-resistant gene) into1-1ras1000 cells, by using the calcium phosphate method,electroporation, lipofection or the like. Other selecting means such asneo-resistant genes or gpt selection may also be used.

3) If the selectable marker is a hygromycin-resistant gene, grow thecells in a medium containing hygromycin to afford hygromycin-resistantcells.

4) Select hygromycin-resistant cells on the basis of invasive/metastaticpotential.

5) Recover non-c-Ha-ras gene fragments containing human gene-specificAlu repeats by using, for example, plaque hybridization.

If a cDNA library prepared with a mammalian cell expression vector wasintroduced, gene fragments can be recovered as episomes (Tatsuka M. etal., Nature 359:333-336, 1992) or using plasmid rescue, PCR or libraryconstruction.

6) Introduce thus recovered gene fragments alone or in mixture into1-1ras1000 cells and examine invasive/metastatic potential.Specifically, for example, phage DNA obtained by plaque hybridizationmay be multiplied for each clone and transfected alone or in mixtureinto 1-1ras1000 cells, and the transfected cells may be injectedsubcutaneously or into the tail vein of nude mice to obtain a phageclone harboring metastatic cells.

7) Isolate nearly full-length CDNA from the CDNA library using a geneconferring invasive/metastatic potential as a probe and determine itsnucleotide sequence. Determine the amino acid sequence on this basis.

8) Screen a human genomic gene library using the cDNA isolated in 7) asa probe to isolate human genomic genes containing all the exons.

9) Integrate the cDNA isolated in 7) into a mammalian cell expressionvector and transfect it into 1-1ras1000 cells to examineinvasive/metastatic potential.

Thus obtained genes can be regarded as genes conferringinvasive/metastatic potential on tumor cells.

Various studies have previously been devoted to screening formetastasis-related genes. For example, Weiberg's group tried expressioncloning of a metastatic gene using NIH3T3 cells with failure (BernsteinS. C. and Weiberg R., Proc. Natl. Acad. Sci. USA 82:1726-1730, 1985).Genes such as nm23 (Leone A. et al., Cell 65:25-35, 1991), CD44(Gunthert U. et al., Cell 65:13-24, 1991), Kai-1 (Dong J. T. et al.,Science 268:884-886, 1995) have been cloned by a process involvingextracting mRNA from highly metastatic tumor cells while separatelyextracting mRNA from less metastatic tumor cells and comparing bothmRNAs to identify mRNA prevailing in highly metastatic tumor cells.Alternatively, an potential metastasis gene (Tiam-1) has been cloned byvirus-insertion mutagenesis (Habets G. G. M. et al., Cell 77:537-549,1994). However, many of genes isolated by such processes were notmetastasis-related genes, indicating that the efficiency of screeningfor metastasis-related genes is still low.

If a gene derived from a tumor cell could be introduced into 1-1ras1000cells of the present invention having tumorigenic potential but lackinginvasive/metastatic potential to confer invasive/metastatic potential onsaid cells, the introduced gene would greatly contribute to theacquisition of invasive/metastatic potential. The above screening methodof the present invention can be used to efficiently identify a geneconferring invasive/metastatic potential on tumor cells, and therefore,it is very useful for investigating the mechanism of metastasis.

In the present invention, the v-src oncogene was actually introducedinto 1-1ras1000 cells to confirm that these cells have acquiredinvasive/metastatic potential. Also v-src conferred invasive/metastaticpotential on the parent strain BALB/c 3T3 A31. These facts suggest thatthe src gene is involved in conferring invasive/metastatic potential.

Further in the present invention, DNA was extracted from tumor celllines having various metastatic potentials and transfected intonon-metastatic 1-1ras1000 cells of the present invention, and thetransformed cells were injected into nude mice to evaluate metastaticpotential. As a result, the transformed cells were found to haveacquired metastatic potential, which was stable and unchanged even aftersubcultures. This demonstrated that 1-1ras1000 cells constitute avaluable screening tool for DNA having tumor metastatic potential.

Thus, the present invention also provides a set of a tumor cell linehaving tumorigenic potential but lacking invasive/metastatic potentialand a tumor cell line derived from the same parent strain and havingboth of tumorigenic potential and invasive/metastatic potential, whichare very useful for investigating the difference between the signalingpathway for acquiring tumorigenic potential and the signaling pathwayfor acquiring invasive/metastatic potential.

The screening method of the present invention can be used todiscriminate benign tumors from malignant tumors, predict theaggressiveness of metastasis or predict organ-specific metastasis.Moreover, genes identified as conferring invasive/metastatic potentialon tumor cells by the screening method of the present invention can beused to obtain metastasis-related proteins that are promising fordeveloping novel diagnostic agents or therapeutic agents for tumors. Thepresent invention has a very wide industrial applicability.

The following examples further explain the present invention in detail,but are not construed as limiting the scope thereof.

EXAMPLES

Strains and plasmids used in the examples of the present invention areat first briefly described below.

Strains

BALB/c 3T3 A31-derived clones 1-1 and 1-13 used as recipient cells inthe present invention were established by Kakunaga (Kakunaga T. et al.,Science 209:505-507, 1980), and are differentially susceptible toradiations—and chemicals—induced neoplastic transformation. Here, earlysubcultures of the original cell stock were used. UV—and MCA(3-methylcholanthrene)—induced transformants (Tatsuka M., Nature359:333-336, 1992) and Ki-MSV (Kirsten murine sarcoma virus)—transformedcells (Kakunaga T., in Omen G. S. et al., (eds.), Genetic Variability inResponses to Chemical Exposure, Cold Spring Harbor Laboratory, New York,1984, pp. 257-274) were also used. All of those transformants weretested to be tumorigenic when subcutaneously injected into nude mice.The negative and positive control cells used in experiments forassessing invasive/metastatic potential were 3Y1 and fos-SR-3Y1-202,respectively (Taniguchi S. et al., Cancer Res. 49:6738-6744, 1989).

All the strains were cultured in EMEM supplemented with 10% FCS in 5%CO₂ at 37° C.

Plasmids

pSV2neo-ras contains the activated c-Ha-ras inserted into the BamHI siteof pSV2neo. pcDsrc contains the v-src oncogene inserted into the EcoRIsite of pcDsrc (Yagi T. et al., Mol. Carcinog. 1:222-228, 1989; Kizakaet al., Mol. Cell. Biol. 9:5669-5675, 1989). The plasmid pHyg carrying ahygromycin-resistant gene was also used.

Example 1

Transformation of BALB/c 3T3 A31 Variant Cells

BALB/c 3T3 A31 variant cells established by Kakunaga, clone 1-1(hereinafter sometimes referred to as A31-1-1) and clone 1-13(hereinafter sometimes referred to as A31-1-13), were examined for theirsusceptibility to in vitro transformation induced by UV-irradiation,MCA-exposure and Ki-MSV carrying the v-Ki-ras oncogene as describedabove.

Focus-Forming Ability of Transformants

A31-1-13 was more susceptible to the chemically and physically inducedneoplastic transformation than A31-1-1, as previously reported (TatsukaM. et al., supra. 1992). Both clones showed similar susceptibility tothe Ki-MSV-induced transformation, as previously reported (Kakunaga T.,supra. 1984). Morphology of the foci differed between both clones butnot with the carcinogens for the induced transformation. The foci werelarger and more aggressive in A31-1-13 than in A31-1-1.

Tumorigenicity and Invasive or Metastatic Potential of Transformants

All the transformants were tested to form tumors when subcutaneouslyinjected into nude mice or newborn BALB/c mice while no tumor was formedwith the parental cells and Mock transfected cells (for example, cellstransformed in the absence of only MCA). Growth rate and latency perioddid not differ significantly between both clones.

Since activated ras oncogenes are known to often induceinvasive/metastatic potential in transformed cells, the transformantscarrying an activated ras oncogene were injected into the tail vein ofnude mice to examine whether they form metastatic lesions in lung.

Throughout the present invention, tumorigenicity and experimentalmetastasis assays were performed in the following manner.

BALB/c nude mice at 6-7 weeks of age were provided. Cells were collectedafter trypsinization and washed with PBS. For tumorigenicity assay, 10⁶cells were injected subcutaneously into the nude mice. For experimentalmetastasis assay, 5×10⁵ cells were injected into the tail vein of 7-8week-old nude mice. The mice injected with ras-transformed cells weresacrificed after 5 weeks and autopsied for metastases. However,preliminary experiments had showed that mice injected withsrc-transformed cells die in 18-24 days as a result of aggressive growthof lung metastatic tumor cells. To avoid this phenomenon, the miceinjected with src-transformed cells were sacrificed and autopsied formetastases at the 2nd week. Metastasized lung nodules were counted afterinsufflation of lung with 15% Indian ink (Wexler H., J. Natl. CancerInst. 36:641-645, 1966).

In some experiments, BALB/c mice of wild type or with suppressed naturalkiller (NK) cells were used. To eliminate NK activity, 200 μl of theanti-asialo GM₁ serum was injected into the tail vein of 7-8 week-oldBALB/c mice for 3 days prior to injection of tumor cells. Theanti-asialo GM₁ serum is known to specifically bind to NK cells tosuppress their function (Kasai M. et al., Eur. J. Immunol. 10:175-180,1980).

The results of tumorigenicity and invasive/metastatic potential obtainedfrom A31-1-1 cells and A31-1-13 cells are shown in the following Table1.

TABLE 1 Metastasis Assay in A31-1-1 and A31-1-13 Cells Transformed withUV, MCA and Ki-MSV No. of No. of mice mice given No. of with nodues Cellline injections nodules (nodules/mouse) Cells Transformed by UV* 1-1UV5† 0 0 1-13UV 5† 0 0 Cells Transformed by MCA* 1-1MCA 5† 0 0 1-13MCA 5†0 0 Cells Transformed by Ki-MSV* 1-1ki-msv1 5† 0 0 1-1ki-msv2 5† 0 01-1ki-msv3 5† 0 0 1-1ki-msv4 5† 0 0 1-1ki-msv5 5† 0 0 1-13ki-msv1 5† 0 01-13ki-msv2 5† 0 0 1-13ki-msv3 5† 0 0 1-13ki-msv4 5† 0 0 1-13ki-msv5 5†0 0 Positive Control 3‡ 324 3 (149,103,72) fos-SR-3Y1-202 NegativeControl 5† 0 0 3Y1 *All of transformants were tumorigenic when 10⁶ cellsof each line were subcutaneously injected into BALB/c # nude mice. †5 ×10⁵ cells of each line were intravenously injected into the tail vein ofBALB/c nude mice. After 5 weeks, the animals were sacrificed andautopsied for lung metastasis. ‡The animals were autopsied at 2 weeks.

As shown from Table 1, no lung invasive/metastatic potential wasobserved in transformed cells. When rat 3Y1 cells transformed with v-srcand v-fos (fos-SR-3Y1-202) were injected as a positive control, a numberof metastatic lesions were found in lung.

Thus, BALB/c 3T3 A31 variant cells, A31-1-1 and A31-1-13, were found tobe susceptible to neoplastic transformation by different kinds ofcarcinogens such as UV irradiation, chemicals and ras oncogenes.Although the transformants were tumorigenic, their invasive/metastaticpotentials examined by experimental invasion/metastasis assay werenegligible.

Example 2

Transfer of the Activated c-Ha-ras Oncogene into BALB/c 3T3 A31 VariantCells

To isolate cells expressing an activated Ras oncoprotein at a highlevel, A31-1-1 and A31-1-13 were transformed by introducing a plasmidcontaining the activated c-Ha-ras (derived from T24 bladder carcinomacells) and a selectable marker neo (pSV2neo-ras). Transformation wasperformed by electroporation (Tatsuka M., et al., Exp. Cell Res.178:154-162, 1988). The transformants were selected in the presence of400 and 1000 μg/ml of G418. Two clones, 1-1 ras and 1-13 ras, wereisolated in the presence of 400 μg/ml of G418 and other two clones,1-1ras1000 and 1-13ras1000, were isolated in the presence of 1000 μg/mlof G418. All the isolated clones had similar properties to those shownby cells transformed with v-Ki-ras. These cells were used for thefollowing experiments.

Southern Blot Analysis of Incorporated Gene

Genomic DNA from each ras-transformed cell was digested with therestriction enzyme SacI and transferred to a nitrocellulose membrane byusing the BIO-DOT SF apparatus (Bio-Rad). The membrane was hybridizedwith the ³²P-labeled ras probe and detected on X-ray film. The copynumber of the introduced gene was determined by densitometry. As aresult, the ras gene introduced into cells was detected as a 3.0-kb SacIband.

Northern Blot Analysis of Transcribed mRNA

Cytoplasmic RNA was prepared by guanidinium thiocyanate-chloroformextraction (Chomczynski P. et al., Anal. Biochem. 162:648-657, 1977).Blotting and hybridization were performed as described by Maniatis etal. (Maniatis T. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1982). The introduced c-Ha-ras andv-src genes were assayed for mRNA using each ³²P-labeled specific probe.As a result, mRNA of ras was detected.

This confirmed that the ras gene had been integrated into the gene ofcells and transcribed into mRNA.

Evaluation of the Copy Number of ras by Slot Blot Analysis

Each 5, 10 or 15 μg of genomic DNA digested with SacI was hybridizedwith the ³²P-labeled SacI/SacI fragment of the c-Ha-ras gene derivedfrom T24 human bladder carcinoma by slot blotting. The results are shownin FIG. 1. The copy number was higher in 1-1ras1000 and 1-13ras1000cells than in the cells selected in the presence of 400 μg/ml of G418(16- and 4-times, respectively).

Immunoblotting Analysis of p21^(ras)

The expression level of Ras oncoprotein was examined by immunoblottingusing an anti-p21^(ras) monoclonal antibody.

Pelleted cells were lysed in Laemmli's sample buffer (Laemmli U. K.,Nature 227:680-685, 1979) by a brief sonication. The proteinconcentration was adjusted to 2 mg/ml. The lysates were heated at 100°C. for 3 min, and loaded onto a 15% polyacrylamide gel. Proteins in thegel were electroblotted onto Durapore filters (Towbin H. et al., Proc.Natl. Acad. Sci. USA 76:4350-4354, 1979) and incubated for 18 h with ananti-p21^(ras) monoclonal antibody, NCC-RAS-004 (Kanai T. et al., Jpn.Cancer Res. 78:1314-1318, 1987). This antibody can detect the p21^(ras)protein transcribed by the c-Ha-ras gene with a point mutation at thecodon 12. During then, specific binding on membranes was blocked withskimmed milk. The proteins transferred on the membranes were furtherincubated with ¹²⁵I-labeled Protein G (Akerstrom B. et al., J. Immunol.135:2589-2592, 1984) for 1.5 h, then autoradiographed on Fuji RX X-rayfilm.

The results are shown in FIG. 2. A rapidly migrating weak bandcorresponds to the endogenous p21^(ras). A slowly migrating band of thep21^(ras) oncoprotein translated from the mutated c-Ha-ras gene(Taparowski E. et al., Nature 300:762-765, 1982) was detectedspecifically in the ras-transformed cells. Such a slowly migrating bandof the p21^(ras) oncoprotein was never detected even when 10-fold excess(50 μl) of the parental cell lysate was electrophoresed on the gel (FIG.2).

Evaluation of Tumorigenicity and Invasive or Metastatic Potential of theCells Transformed with c-Ha-ras and v-src

To examine whether or not an increased expression level of the p21^(ras)oncoprotein affects the invasive/metastatic potential of transformants,the cells transformed with c-Ha-ras and v-src were tested for theirtumorigenicity and invasive/metastatic potential. The results are shownin the following Table 2.

TABLE 2 Tumorigenicity and Invasive or metastatic Potential in A31-1-1and A31-1-13 Cells Transformed with c-Ha-ras and v-src OncogenesTumorigenicity Metastatic potential No. of No. tumor bearing of mice No.of mice mice/No. of given No. of with nodules Cell line injected miceinjections nodules (nodules/mouse) Parental Variant Cells or CellsTransfected with Empty Vector 1-1 0/5* 5‡ 0 0 1-13 0/5 5‡  0 0 1-1neo ND3‡  0 0 1-13neo ND 3‡  0 0 Cells Transfected with Mutated c-Ha-ras1Oncogene 1-1ras4 3/3† 5‡  0 0 1-1ras1000 3/3† 5‡  0 0 1-13ras2 3/3† 5‡ 0 0 1-13ras1000 3/3† 5‡  0 0 Cells Transfected with v-src Oncogene1-1src, 313† 3§ 154 3 (58,48,48) pooled cells 1-1src,clone1 ND 3§ 286 3(126,89,71) 1-1src,clone2 ND 3§ 213 3 (94,67,52) 1-1src,clone3 ND 3§ 3163 (118,116,82) 1-13src, 3/3† 3§ 172 3 (69,53,50) pooled cells 1-13src,ND 3§ 203 3 (85,77,41) clone1 1-13src, ND 3§ 342 3 clone2 (134,105,103)1-13src, ND 3§ 265 3 (104,92,69) clone3 Additional Transfection withv-src Oncogene or Empty Vector to 1-1ras1000 Cells 1-1ras1000hyg 3/3† 6‡0 0 1-1ras1000src 3/3† 9§ 1227 9(196,165,146, 138,130,124 116,112,100)*Mice injected subcutaneously with 1 × 10⁶ of each cell variant weretumor-free up to 10 weeks # after injection. †Cells from ras-andsrc-transfectants gave rise to tumors within 1-2 weeks. In all ofinjected # animals, tumor weight was 9-10 g after 3 weeks. ‡The animalswere autopsied at 5 weeks. §The animals were autopsied at 2 weeks.

As shown from Table 2, the increased expression level of the p21^(ras)oncoprotein did not affect at all the invasive/metastatic potential oftransformants.

This revealed that the invasive/metastatic phenotype of theras-transformed cells was very stable and was hardly changed during invitro cultivation.

Example 3

Invasive or Metastatic Potential of BALB/c 3T3 Variant Cells can beInduced by a Viral Oncogene v-src

A viral oncogene, v-src, has a strong transforming activity and caninduce invasive/metastatic potential simultaneously, depending on thetype of the recipient cell. Thus, the effect of v-Src oncoprotein on theparental BALB/c 3T3 A31 variant cells as well as on the ras-transformedcells was examined by the transfection with this oncogene.

Upon transfection with v-src, the parental cells (BALB/c 3T3 A31 cells)formed foci in vitro, indicating that the v-src gene itself has theactivity of transforming cells. The shape of the focus-formingtransformed parental cells was not so different from that of the cellstransformed with the mutated ras gene.

Then, the transformants were confirmed to contain the transfected v-srcgene as a 2.9-kb EcoRI fragment by Southern blotting and a functionalexpression of v-Src oncoprotein was also confirmed as follows.

Immunoblotting Analysis and Tyrosine Kinase Assay of p60^(src)

The expression level of v-src gene products in src-transformants wereexamined by immunoblotting analysis of p60^(src) using anti-p60^(src)serum specific to Src protein (Cooper J. A. et al., J. Virol.48:752-764, 1983). The src-transformants were also examined forphosphorylation of Src protein by the kination assay as described byJove et al. (Jove R. et al., J. Virol. 60:849-857, 1986).

As a result, it was difficult to distinguish v-Src from the endogenousc-Src by immunoblotting analysis (FIG. 3A). However, an enhancedautophosphorylation of the p60^(src) was observed in the transformedcells by kination assay following immunoprecipitation experiment (usinganti-p60^(src) serum) (FIG. 3B).

src-Transformed Cells Have Acquired Tumorigenicity and Invasive orMetastatic Potential

A pooled fraction of the src-transformed cells derived from A31-1-1 andA31-1-13 efficiently formed tumors within 1-2 weeks after injection, andthe latency period was the same as that of the ras-transformed cells.The pooled transformants and each transformed clone (1-1src clones 1 to3, 1-13src clones 1 to 3, and 1-1ras1000src) formed a number of lungmetastases by the experimental invasion/metastasis assay (Table 2).

Thus, a clear difference was observed between the activated ras- andv-src-transformed cells in their invasive/metastatic potential analyzedby the experimental invasion/metastasis assay. In order to examinewhether or not this difference results from the sensitivity of theras-transformed cells to nude mice, metastatic potentials of theras-transformed 1-1ras1000 cells and src-transformed 1-1 src cells werecompared using wild-type and NK-suppressed BALB/c mice instead of nudemice. This yielded the same results in either wild-type or NK-suppressedmice (FIG. 4), indicating that the above difference in metastaticpotential is not due to the sensitivity to NK activity in nude mice.

Example 4

In vitro Invasiveness and Cell Motility of the ras- and src-TransformedCells

In vitro cell motility and invasiveness are known to be closely relatedto the invasive/metastatic potential of tumor cells. An invasive abilityof cells was analyzed by the method of Albini et al. (Albini A. et al.,Cancer Res. 47:3239-3245, 1987) using a modified Boyden chamber fittedwith a MATRIGEL-coated filter. As a result, v-src transformed cellsA31-1-1 and A31-1-13 showed invasion indices 4-5 times higher than thoseof the ras-transformed cells (FIG. 5A). The invasiveness was notaffected by an increased expression level of the mutated Rasoncoprotein.

Cell motility was measured on MATRIGEL-coated dishes usingcomputer-aided digital image analysis (Tatsuka M. et al., Exp. Cell Res.185:342-352, 1989) and indicated by the motility index. As a result, themotility indices of the src-transformed cells were also 6-7 times higherthan those of the ras-transformed cells (FIG. 5B). This suggests thatv-Src oncoprotein induces various additional cellular phenotypes thatare not induced by the activated Ras protein, and consequently thesrc-transformants become invasive/metastatic.

Example 5

Induction of Metastatic Potential by DNA Derived from Human Tumor Cells

DNA extracted from various human metastatic tumor cells was transfectedinto non-metastatic 1-1ras1000 cells to examine induction of metastaticpotential.

A. Experimental Procedures

(1) Extraction of DNA

The following human tumor cell lines were used as experimental materialsto extract whole genomic DNA according to the method of Maniatis et al.(Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1982).

A204 (ATCC HTB-82): rhabdomyosarcoma;

Malme-3M (ATCC HTB-64): lung-metastasized malignant melanoma;

Mewo (JCRB0066): malignant melanoma;

SW480 (ATCC CCL-228): colon adenocarcinoma.

These cells were obtained from American Type Culture Collection (ATCC)or Japanese Collection of Research Biosources (JCRB).

The size of each DNA extracted from these cell lines exceeded 20-90 kb,as determined by pulsed field electrophoresis.

(2) DNA Transfection

DNA transfection was performed by the calcium phosphate method (Graham,F. L. and Van Der EB, A. J., Transformation of rat cells by DNA of humanadenoma 5, Virology, 54:536-539, 1973). Prior to transfection, 5×10⁵1-1ras1000 cells were seeded in dishes of 100 mm in diameter. Eachgenomic DNA sample prepared by mixing 1 mg of genomic DNA with 100 μg ofpSV2gptDNA (dominant selectable marker) was precipitated and added in 20dishes. After 4 hours, cells were shocked with 15% glycerol for 60 sec.Cells were grown for 16 h in Eagle's MEM supplemented with 10% fetalbovine serum. Then, a selective medium containing xanthine (250 μg/ml),hypoxanthine (15 μg/ml), thymidine (10 μg/ml), aminopterin (2 μg/ml) andmycophenolic acid (MPA: 25 μg/ml) was added to each dish. MPA-resistantcolonies which appeared on dishes were pooled for further experiments.

(3) Evaluation of Metastatic Potential

Male BALB/c nude mice were obtained at 6-7 weeks of age. MPA-resistantcells were collected by trypisinization. The pooled cells from eachtransfection were washed with Hanks' solution and 5×10⁵ cells wereinjected into the tail vein of 7-8 week-old nude mice. The animals weresacrificed after 5 weeks and autopsied. Cells were obtained by cultureof minced lung. If a metastatic lesion was formed in lung, a colonyappeared upon culture on an MPA-selective medium. Normally, a colony wasgenerated from each focus metastasized to the lung of one mouse. Thenumber of colonies was counted and all the colonies from each mouse wereused for a second round of transfection.

B. Results

MPA-resistant colonies incorporating DNA from the cell line A204,Malme-3M, Mewo or Sw480 metastasized to lung (Table 3). MPA-resistantcolonies recovered from the lung with metastases were pooled andcultured, and then reinjected into mice. As a result, all the coloniesmetastasized to lung (Table 3).

A pooled strain of 1-1ras1000 cells incorporating DNA of SW480 was usedfor further transfection experiments. When DNA extracted from thisstrain was retransfected into 1-1ras1000 (second transfection), thosecells metastasized to lung. When the same procedure was repeated again(third transfection), lung metastasis was observed. The number ofcolonies metastasized to lung increased with an increase of rounds oftransfection (Table 3). Transformed cells were stable with no change inmetastatic potential after at least 5 passages in normal medium.

TABLE 3 No. of mice given No. of colonies DNA tranfected i.v.administration observed per mouse First transfection 1-1ras1000 50,0,0,0,0 A204 5 3,4,12,15,18¹ Malme-3M 5 0,5,6,8,9² Mewo 5 0,0,0,1,2³SW480 5 0,3,5,6,8⁴ Second transfection 1-1ras1000 6 0,0,0,0,0,11-1ras1000 SW480-T1 6 15,20,24,34,48,63⁵ Third transfection 1-1ras1000 40,0,0,0 1-1ras1000 SW480-T2 4 23, >200, >200, >200 ¹Metastasis occurredwhen 10⁵ cells recovered from 18 colonies were reinjected into the tailvein of # nude mice. ²Metastasis occurred when 10⁵ cells recovered from9 colonies were reinjected into the tail vein of # nude mice.³Metastasis occurred when 10⁵ cells recovered from 2 colonies werereinjected into the tail vein of # nude mice. ⁴Cells recovered from 8colonies (1-1ras1000 SW480-T1) were used for a second round oftransfection. ⁵Cells recovered from 63 colonies (1-1ras1000 SW480-T2)were used for a third round of transfection.

What is claimed is:
 1. A tumor cell obtained by transfecting a BALB/c3T3 A31 cell with an oncogene of the ras family, wherein said cell hastumorigenic potential but lacks invasive or metastatic potential, andwherein said tumorigenic potential and said lack of invasive ormetastatic potential have been conferred by introduction of the oncogeneof the ras family, and without co-transfecting with another oncogene. 2.The tumor cell according to claim 1, wherein the oncogene of the rasfamily is the c-Ha-ras gene.
 3. The tumor cell according to claim 1,which is 1-1 ras 1000 (accession number FERM BP-5406).
 4. A method forpreparing the tumor cell according to claim 1, comprising providing aBALB/c 3T3 A31 clone; and introducing an oncogene of the ras family intosaid BALB/c 3T3 A31 clone.
 5. The tumor cell according to claim 1, whichis a BALB/c 3T3 A31-1-1 or a BALB/c 3T3 A31-1-13 cell.
 6. The tumor cellof claim 1, wherein said oncogene is an activated oncogene.
 7. The tumorcell of claim 1, wherein said oncogene is an activated c-Ha-ras.
 8. Themethod according to claim 4, wherein the BALB/c 3T3 A31 clone is BALB/c3T3 A31-1-1 or BALB/c 3T3 A31-1-13.
 9. The method according to claim 4,wherein the oncogene of the ras family is the c-Ha-ras gene.
 10. Themethod according to claim 4, wherein the tumor cell is 1-1 ras 1000(Accession No. FERM BP-5406).
 11. A screening method for genes havingthe property of conferring invasive or metastatic potential, whichcomprises transfecting DNA of a tumor tissue obtained from the surfaceor inside of a mammal or of a tumor cell line into a tumor cell havingtumorigenic potential but lacking invasive or metastatic potential,isolating cells having acquired invasive or metastatic potential andextracting DNA therefrom, wherein said tumor cell is obtained from aBALB/c 3T3 A31 cell and wherein said tumorigenic potential has beenconferred by an oncogene of the ras family.
 12. The method according toclaim 11, wherein the DNA is a genomic gene of a metastatic tumor cellor a cDNA library prepared with a mammalian cell expression vector. 13.The method according to claim 12, wherein the tumor cell transfectedwith DNA is 1-1 ras 1000 (Accession No. FERM BP-5406).
 14. A tumor cellobtained from a BALB/c 3T3 A31 cell transfected with an oncogene of theras family and a src oncogene, wherein said cell has tumorigenicpotential and invasive or metastatic potential.